Structure of the Cytoplasmic Region of PelD, a Degenerate Diguanylate Cyclase Receptor That Regulates Exopolysaccharide Production in Pseudomonas aeruginosa

• Published in: The Journal of biological chemistry Vol. 287; no. 28; pp. 23582 - 23593
• Main Authors: Whitney, John C., Colvin, Kelly M., Marmont, Lindsey S., Robinson, Howard, Parsek, Matthew R., Howell, P. Lynne
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.07.2012; American Society for Biochemistry and Molecular Biology
• Subjects: 59; Amino Acid Motifs - genetics; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Binding, Competitive; Biofilm; Biofilms; Blotting, Western; Crystal Structure; Crystallography, X-Ray; Cyclic Di-GMP; Cyclic GMP - analogs & derivatives; Cyclic GMP - metabolism; Cyclic Nucleotides; Extracellular Matrix; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microbial Pathogenesis; Microbiology; Models, Molecular; Molecular Sequence Data; Mutation; PEL Polysaccharide; Polysaccharide; Polysaccharide-Lyases - chemistry; Polysaccharide-Lyases - genetics; Polysaccharide-Lyases - metabolism; Polysaccharides, Bacterial - metabolism; Protein Structure, Secondary; Protein Structure, Tertiary; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - metabolism; Pseudomonas aeruginosa - physiology; X-ray Crystallography; Crystal Structure; Extracellular Matrix; X-ray Crystallography; PEL Polysaccharide; Biofilm; Cyclic Di-GMP; Cyclic Nucleotides; Polysaccharide; Microbial Pathogenesis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.375378

High cellular concentrations of bis-(3′,5′)-cyclic dimeric guanosine mono-phosphate (c-di-GMP) regulate a diverse range of phenotypes in bacteria including biofilm development. The opportunistic pathogen Pseudomonas aeruginosa produces the PEL polysaccharide to form a biofilm at the air-liquid interface of standing cultures. Among the proteins required for PEL polysaccharide production, PelD has been identified as a membrane-bound c-di-GMP-specific receptor. In this work, we present the x-ray crystal structure of a soluble cytoplasmic region of PelD in its apo and c-di-GMP complexed forms. The structure of PelD reveals an N-terminal GAF domain and a C-terminal degenerate GGDEF domain, the latter of which binds dimeric c-di-GMP at an RXXD motif that normally serves as an allosteric inhibition site for active diguanylate cyclases. Using isothermal titration calorimetry, we demonstrate that PelD binds c-di-GMP with low micromolar affinity and that mutation of residues involved in binding not only decreases the affinity of this interaction but also abrogates PEL-specific phenotypes in vivo. Bioinformatics analysis of the juxtamembrane region of PelD suggests that it contains an α-helical stalk region that connects the soluble region to the transmembrane domains and that similarly to other GAF domain containing proteins, this region likely forms a coiled-coil motif that mediates dimerization. PelD with Alg44 and BcsA of the alginate and cellulose secretion systems, respectively, collectively constitute a group of c-di-GMP receptors that appear to regulate exopolysaccharide assembly at the protein level through activation of their associated glycosyl transferases. Background: Binding of c-di-GMP to PelD regulates the biosynthesis of PEL exopolysaccharide. Results: Apo and c-di-GMP complexed structures of the cytoplasmic region of PelD have been determined. Conclusion: PelD contains a GAF domain and a degenerate GGDEF domain. Dimeric c-di-GMP binds at a conserved allosteric inhibition site commonly found in diguanylate cyclases. Significance: This is the first structural characterization of a degenerate GGDEF domain c-di-GMP receptor.